The present invention relates to cytotactin proteins, polypeptides, antibodies and other cytotactin derivatives useful in the mediation of neuronal attachment and enhancement of the outgrowth of neurites, as well as to methods of using same. Methods of making the disclosed proteins, polypeptides, antibodies, derivatives and related compositions, which have a variety of diagnostic and therapeutic applications, are also disclosed.
Cytotactin (CT) is a multidomain extracellular matrix (ECM) protein which plays a role in cell migration, proliferation, and differentiation during development (Crossin, et al., J. Cell Biol. 102: 1917-1930(1986); Prieto, et al., J. Cell Biol. 111: 685-698 (1990)), which may be controlled by other developmentally important genes. The restricted spatiotemporal expression of cytotactin that results from its developmental regulation is tightly linked to a number of cellular primary processes, including adhesion (Grumet, et al., Proc. Natl. Acad. Sci. USA 82: 8075-8079 (1985)), migration (Chuong et al., J. Cell Biol. 104: 331-342 (1987); Halfter, et al., Dev. Biol. 132: 14-25 (1989); Tan, et al., PNAS USA 84: 7977-7981 (1987)), proliferation (Chiquet-Ehrismann, et al., Cell 53: 383-390 (1988); Crossin, PNAS USA88: 11403-11407 (1991)), differentiation (Mackie, et al., J. Cell Biol. 105: 2569-2579 (1987)), epithelial-mesenchymal interactions (Aufderheide, et al., J. Cell Biol. 105: 2341-2349 (1988); Aufderheide, et al., J. Cell Biol. 105: 599-608 (1987)), and cell death (Williamson, et al., Embryonic Develop. Morphol. 209: 189-202 (1991)).
Cytotactin, which is also known as tenascin (TN) (Chiquet-Ehrismann, et al., Cell 47: 131-139 (1986)), J1 2201200 (Kruse, et al., Nature 316: 146-148 (1985)), hexabrachion (Erickson, et al., Nature 311: 267-269 (1984); Gulcher, et al., PNAS USA 86: 1588-1592 (1989)), the glioma-mesenchymal extracellular matrix protein (Bourdon, et al., Cancer Res. 43: 2796-2805 (1983)), and myotendinous antigen (Chiquet et al., J. Cell Biol. 98: 1926-1936 (1984)), exists in at least three isoforms generated by alternative splicing (Zisch, et al., J. Cell Biol. 119: 203 (1992)). The three known chicken CT isoforms, which are composed of polypeptides having molecular weights of 190, 200, and 220 kD have been isolated from chicken brain (Grumet, et al., PNAS USA 82: 8075-8079 (1985)); relative to the 190 kD isoform, the 200 kD form contains one, and the 220 kD form contains three, additional fn type III domains (Zisch, Id, (1992)). The CT found in other species, including human and murine CT, for example, exists in a variety of isoforms as well.
As noted, variation in the polypeptide structure arises from alternative splicing of transcripts from a single gene (Jones, et al., PNAS USA 85: 2186-2190 (1988); Jones, et al., PNAS USA 86: 1905-1909 (1989); Spring, et al., Cell 59: 325-334 (1989)). The polypeptides are disulfide-linked to form a multimeric structure (Grumet, et al., PNAS USA 82: 8075-8079 (1985); Hoffman, et al., J. Cell Biol. 106: 519-532 (1988)). Electron microscopy of the rotary-shadowed molecule has revealed a characteristic six-armed structure, called a hexabrachion (Erickson, et al., Nature 311: 267-269 (1984); Erickson, et al., Adv. Cell Biol. 2: 55-90 (1988)), in which six polypeptides are linked through disulfide bonds at their aminotermini.
The sequence of cytotactin reveals a multidomain structure (Jones, et al., PNAS USA 86: 1905-1909 (1989); Spring, et al., Cell 59: 325-334 (1989)) with homologies to three other protein families. The amino-terminal portion contains the cysteine involved in interchain disulfide bonding, followed by an array of 13 repeats of 31 amino acids in length that resemble those found in epidermal growth factor (EGF). These EGF-like repeats are followed by a variable number of repeats similar to fibronectin type III repeats. In the chicken, cytotactin polypeptides contain between 8 and 11 type III repeats as a consequence of alternative RNA splicing. Different variants have been shown to be expressed preferentially at certain times and anatomical sites during development (Prieto, et al., J. Cell Biol. 111: 685-698 (1990)) and they may have different binding or morphogenic functions (Kaptony, et al., Development (Camb.) 112: 605-614 (1991); Matsuoka, et al., Cell Differ. 32: 417-424 (1990); Murphy-Ullrich, et al., J. Cell Biol. 115: 1127-1136 (1991)).
More recently, it has been shown that the third fibronectin type III (CTfn3) repeat can mediate RGD-dependent cell attachment via integrins xcex1vxcex23 and xcex1vxcex26 and that the whole molecule bound to a xcex21 integrin but the binding site was not determined. The carboxy-terminal portion of cytotactin is homologous to the distal domain of the xcex2 and xcex3 chains of fibrinogen and contains a putative Ca2+ binding site.
Early studies of cell attachment to cytotactin-coated surfaces suggested that multiple modes of binding to the molecule existed. For example, fibroblasts bind both to intact cytotactin and to a chymotryptic fragment derived from the carboxy-terminal end of the protein (Friedlander, et al., J. Cell Biol. 107: 2329-2340 (1988)). These binding activities are inhibitable by peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD) and by antibodies to specific regions of the cytotactin protein. In contrast to their rounded cell morphology on intact cytotactin, cells exhibit a spread morphology on the chymotryptic fragment. Using a variety of recombinant fragments of cytotactin, a smaller region of the molecule has been identified as a cell binding site, but no spreading was observed (Spring, et al., Cell 59: 325-334 (1989)).
In these studies, a fragment in the amino-terminal region containing the EGF domains appeared to prevent cell binding to other substrates. Together, these observations suggested that at least two binding activities are present in intact cytotactin, one in the carboxy-terminal half of the protein, mediating cell attachment and flattening, and one in the amino-terminal portion, responsible for so-called anti-adhesive effects (Spring, et al., Cell 59: 325-334 (1989)) and rounding of cells exposed to the molecule (Chiquet-Ehrismann, et al., Cell 47: 131-139 (1986); Friedlander, et al., J. Cell Biol. 107: 2329-2340 (1988)). Studies on the effects of cytotactin on neural attachment and neurite outgrowth have suggested at least one additional interactive site on the molecule based on antibody inhibition studies (Crossin, et al., Exp. Neurol. 109: 6-18 (1990); Faissner, et al., Neuron 5: 627-637 (1990); Grierson, et al., Dev. Brain Res. 55: 11-19 (1990); Husmann, et al., J. Cell Biol. 116: 1475-1486(1992); Lochter, et al., J. Cell Biol. 113: 1159-1171 (1991); Wehrle, et al., Development (Camb.) 110: 401-415 (1990)).
We have now unambiguously identified the regions of CT responsible for its ability to promote or to inhibit neurite outgrowth, as well as the regions primarily responsible for cell attachment and spreading. Understanding which regions of this complex protein are responsible for these various functions is essential to determine how the protein may affect neural development and regeneration. One working hypothesis is that the inhibition and promotion of neurite outgrowth may be mapped to specific domains of the protein and may be modulated by other CT binding proteins in the ECM. Fusion proteins have now been generated in the pGEX expression system comprising almost the entire linear structure of the protein and have now been expressed in bacteria. Other new constructs comprising portions of CT, some in unique combinations, are also disclosed herein.
Using these bacterially-generated fusion proteins, smaller domains within the CT protein (e.g., CTfn3) have now been identified that have the ability to promote neurite outgrowth. Another major contribution of the within-disclosed invention is the contribution to the understanding of the conditions under which CT facilitates or inhibits neurite outgrowth and the description of reagents useful in therapeutic interventions to improve neural regeneration.
Therefore, in one embodiment, the present invention contemplates a cytotactin (CT) polypeptide substantially homologous to at least a portion of the protein identified as SEQ ID NO 2 herein (human cytotactin encoded by SEQ ID NO 1), wherein the polypeptide comprises not more than 250 amino acid residues in length. In another variation, the CT polypeptide is substantially homologous to at least a portion of the protein identified as SEQ ID NO 4 herein (chicken cytotactin encoded by SEQ ID NO 3). In various embodiments, the CT polypeptides are capable of stimulating neuronal cell attachment, cell elongation, cell growth, neurite outgrowth, or a combination of the foregoing. In an alternative embodiment, a polypeptide of the present invention is capable of stimulating cell attachment to a substrate, or it may be incorporated into a bioabsorbable matrix.
The invention further contemplates a polypeptide substantially homologous to at least a portion of the protein identified as SEQ ID NO 2 or SEQ ID NO 4 herein (respectively human and chicken cytotactin respectively encoded by SEQ ID NO 1 and SEQ ID NO 3), wherein the polypeptide comprises not more than 250 amino acid residues in length, and wherein the polypeptide comprises a fusion of two or more segments of the protein identified as SEQ ID NO 2 or SEQ ID NO 4 herein. In various alternative embodiments, the polypeptide has an amino acid residue sequence selected from the group consisting of SEQ ID NO 5 (human cytotactin); SEQ ID NO 6 (mouse cytotactin); SEQ ID NO 7 (chicken cytotactin); SEQ ID NO 8 (human cytotactin); SEQ ID NO 9 (mouse cytotactin); and SEQ ID NO 10 (chicken cytotactin). In other variations, the polypeptide is selected from the group consisting of CTfn3, CTfn6, and CTfn3-6.
The present invention also contemplates a cytotactin (CT) polypeptide substantially homologous to at least a portion of the protein identified as SEQ ID NO 4 herein, wherein the polypeptide comprises not more than 250 amino acid residues in length. The various alternative embodiments and applications described hereinabove with respect to SEQ ID NO 2 are also contemplated with regard to SEQ ID NO 4.
In yet another embodiment, the present invention contemplates a biological material comprising a bioabsorbable matrix and an effective amount of a pharmacologically active agent capable of affecting cell attachment, cell growth, or neurite outgrowth. In one variation, the biological material further comprises a collagen gel. In another variation, the agent comprises a cytotactin derivative. In alternative embodiments, the cytotactin derivative comprises human cytotactin (SEQ ID NO 2) or chick cytotactin (SEQ ID NO 4).
In yet another variation pertaining to biological materials of the present invention, the cytotactin derivative comprises one or more cytotactin polypeptides. The invention further contemplates that the cytotactin polypeptides are selected from the group consisting of SEQ ID NO 5; SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 8; SEQ ID NO 9; and SEQ ID NO 10. Alternatively, the cytotactin polypeptides are selected from the group consisting of CTfn3, CTfn6, and CTfn3-6. Another embodiment contemplates that the cytotactin derivative comprises an anti-(CT idiotype) antibody.
The invention further contemplates that the matrix comprises a bioabsorbable biopolymer. In various embodiments, the biopolymer comprises one or more macromolecules selected from the group consisting of collagen, elastin, fibronectin, vitronectin, laminin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparin sulfate, heparin, fibrin, cellulose, gelatin, polylysine, echinonectin, entactin, thrombospondin, uvomorulin, biglycan, decorin, and dextran. In another disclosed variation, the matrix further includes a substructure comprising freeze dried sponge, powders, films, flaked or broken films, aggregates, microspheres, fibers, fiber bundles, or a combination thereof. In yet another embodiment, the matrix further includes a solid support selected from the group consisting of a prosthetic device; a porous tissue culture insert; an implant; and a suture.
The within-disclosed invention also contemplates antibody compositions. In one variation, an antibody composition comprises antibody molecules capable of inhibiting neurite outgrowth, wherein the antibody molecules immunoreact with a CT polypeptide substantially homologous to at least a portion of the protein identified as SEQ ID NO 2 herein. In another variation, the CT polypeptide is substantially homologous to at least a portion of the protein identified as SEQ ID NO 4 herein.
Another embodiment contemplates that the antibody molecules also immunoreact with cytotactin. In other variations, the antibody molecules are monoclonal or polyclonal. In one disclosed embodiment, the CT polypeptide is selected from the group consisting of SEQ ID NO 5; SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 8; SEQ ID NO 9; and SEQ ID NO 10. In another variation, the CT polypeptide is selected from the group consisting of CTfn3, CTfn6, and CTfn3-6.
Another antibody composition contemplated herein comprises anti-(CT idiotype) antibody molecules capable of stimulating neurite outgrowth. In one embodiment, the anti-(CT idiotype) antibody molecules have an activity substantially similar to that of a polypeptide substantially homologous to at least a portion of a protein identified as SEQ ID NO 2 or SEQ ID NO 4 herein, wherein the polypeptide comprises not more than 250 amino acid residues in length. In another embodiment, the anti-(CT idiotype) antibody molecules have an activity substantially similar to that of a polypeptide selected from the group consisting of CTfn3, CTfn6, and CTfn3-6. In one variation, the anti-(CT idiotype) antibody molecules are monoclonal. In another, the antibodies are humanized.
The present invention also discloses methods for preparing solid supports useful in promoting neuronal cell growth and elongation (and/or neurite outgrowth), comprising coating or impregnating the solid support with a biological material including a cytotactin derivative capable of promoting the growth and elongation. In one disclosed variation, the biological material comprises a bioabsorbable biopolymer. In another variation, the solid support is selected from the group consisting of a porous tissue culture insert; a prosthetic device; an implant; and a suture.
In another embodiment, the solid support comprises a bioabsorbable biopolymer. In alternative variations, the biopolymer comprises one or more macromolecules selected from the group consisting of collagen, elastin, fibronectin, vitronectin, laminin, hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparin sulfate, heparin, fibrin, cellulose, gelatin, polylysine, echinonectin, entactin, thrombospondin, uvomorulin, biglycan, decorin, and dextran. In another embodiment, the biological material further comprises at least one attachment factor. Another variant of the disclosed method contemplates that the attachment factor is selected from the group consisting of collagen (all types), fibronectin, gelatin, laminin, polylysine, vitronectin, cytotactin, echinonectin, entactin, thrombospondin, uvomorulin, biglycan, chondroitin sulfate, decorin, dermatan sulfate, heparin, and hyaluronic acid.
The present invention also encompasses a variety of diagnostic and therapeutic assays and kits. In one embodiment, an assay kit for the detection of tumors comprises in an amount sufficient to conduct at least one assay, an anti-cytotactin antibody. Further components in various embodiments include labeling means, samples of CT protein or polypeptide, anti-(CT idiotype) antibodies, and other CT derivatives, all in amounts sufficient to conduct at least one assay.
The invention also contemplates various compounds and compositions useful in the detection or inhibition of metastasis or angiogenesis. One embodiment contemplates a site-specific anti-CT antibody capable of inhibiting metastasis in an individual. Another discloses a polypeptide capable of inhibiting metastasis and angiogenesis in an individual via modulating cell attachment to cytotactin.
The present invention also discloses various methods of detecting tumors. One method comprises obtaining a fluid or tissue sample from an individual; admixing the sample with a predetermined amount of an anti-cytotactin antibody to form an admixture; maintaining the admixture for a time period sufficient to allow the antibody to immunoreact with any cytotactin or fragments thereof in the sample, to form an immunoreaction product; assaying for the presence of the immunoreaction product; and comparing the amount of immunoreaction product assayed with a control, thereby determining whether an excessive amount of cytotactin is present in the sample.
Cell culture systems and methods are also contemplated herein. In one embodiment, a cell culture system comprising a substrate with a cell adhesion factor attached thereto is disclosed. In another, the adhesion factor comprises a CT derivative. Yet another discloses that the CT derivative is selected from the group consisting of SEQ ID NO 2; SEQ ID NO 4; SEQ ID NO 5; SEQ ID NO 6; SEQ ID NO 7; SEQ ID NO 8; SEQ ID NO 9; and SEQ ID NO 10.
A method of inhibiting cytotactin binding to neuronal cells in a patient is also disclosed, comprising administering to the patient a physiologically tolerable composition comprising a therapeutically effective amount of a CT derivative. In one alternative embodiment, the CT derivative is an antibody; in another, it is an anti-(CT idiotype) antibody. In still another variation, the therapeutically effective amount is an amount sufficient to produce an intravascular concentration of antibody in the blood of the patient in the range of about 0.1 to 100 xcexcg/ml. Yet another variation contemplates that the CT derivative is a CT polypeptide substantially homologous to at least a portion of the protein identified as SEQ ID NO 2 herein.
In various disclosed embodiments, a therapeutically effective amount is an amount sufficient to produce an intravascular concentration of CT polypeptide in the blood of the patient in the range of about 0.1 to 100 micromolar. According to various embodiments, the neuronal cells are fibroblasts or ganglion cells.
Various compositions are also encompassed herein. In one embodiment, a composition comprises a therapeutically effective amount of a CT derivative in a pharmaceutically acceptable excipient, wherein the effective amount is an amount sufficient to inhibit cytotactin binding to neuronal cells. In another variation, the CT derivative is a CT polypeptide substantially homologous to at least a portion of the protein identified as SEQ ID NO 2 herein. Another embodiment contemplates that the effective amount is at least 0.1 weight percent of CT derivative per total weight of the composition. In various disclosed embodiments, the CT derivative is an anti-CT antibody or an anti-(CT idiotype) antibody.
The invention further contemplates methods of assaying the amount of cytotactin in a fluid sample. One such method comprises the steps of (a) admixing a fluid sample with an anti-CT antibody to form an immunoreaction admixture; (b) maintaining the admixture for a time period sufficient to form a CT-containing immunoreaction product in a solid phase; and (c) determining the amount of product formed in step (b). In another embodiment, the antibody is a monoclonal antibody; in yet another, the antibody is capable of immunoreacting with CTfn3 or CTfn6, or both.
In an alternative embodiment, the determining step (c) comprises the steps of (1) admixing the CT-containing immunoreaction product in the solid phase with a second antibody to form a second immunoreaction admixture having a liquid phase and a solid phase, the second antibody having the ability to immunoreact with the CT-containing immunoreaction product; (2) maintaining the second reaction admixture for a time period sufficient for the second antibody to immunoreact with the CT-containing immunoreaction product and form a second immunoreaction product in the solid phase; and (3) determining the amount of the second antibody present in the second immunoreaction product, thereby determining the amount of CT-containing immunoreaction product formed in step (c).
The present invention also discloses a competition assay method for assaying the amount of cytotactin in a fluid sample, comprising the steps of (a) forming a competition immunoreaction admixture by admixing a vascular fluid sample with (1) an anti-CT antibody composition containing antibody molecules that immunoreact with cytotactin and with a CT polypeptide substantially homologous to at least a portion of the protein identified as SEQ ID NO 2 herein, wherein the antibody molecules are attached to a solid matrix, such that the competition immunoreaction admixture has both a liquid and a solid phase; and (2) a polypeptide immunoreactive with the antibody, wherein the polypeptide is labeled; (b) maintaining the competition immunoreaction admixture for a time period sufficient to form a labeled immunoreaction product in the solid phase; and (c) determining the amount of labeled immunoreaction product formed in step (b), thereby determining the amount of cytotactin present in the sample. In one variation, the antibody is a monoclonal antibody. In alternative embodiments, the antibody is capable of immunoreacting with CTfn3, CTfn6, or both.
Finally, another preferred embodiment of the invention relates to polynucleotides which encode the above noted cytotactin proteins and polypeptides, and to polynucleotide sequences which are complementary to these polynucleotide sequences. Complementary polynucleotide sequences include those sequences which hybridize to the polynucleotide sequences of the invention under stringent hybridization conditions. Methods of making the various proteins, polypeptides, and other CT derivatives disclosed herein are also inventions disclosed herein.